Randall J Brezski

Janssen Research & Development, LLC, Raritan, New Jersey, United States

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Publications (18)98.93 Total impact

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    ABSTRACT: We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab') 2 fragments, but not for full-length IgG or for closely-related F(ab') 2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab') 2 fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab') 2 fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab') 2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab') 2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab') 2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab') 2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.
    mAbs 08/2014; 6(5). · 5.28 Impact Factor
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    ABSTRACT: The functional role of human anti-hinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 03/2014; · 3.34 Impact Factor
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    ABSTRACT: Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. A necessary step to engineer the most appropriate properties for a particular therapeutic area requires an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly-adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen-binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease-resistance, but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions, while still retaining protease-resistance. By identifying mutations that can restore either complement or FcγR-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.
    Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
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    ABSTRACT: The Fc variant of IgG2, designated as IgG2, was engineered with V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement protein and consequently, immune effector functions. IgG2σ was compared to other previously well-characterized Fc 'muted' variants, including aglycosylated IgG1, IgG2m4 (H268Q/V309L/A330S/P331S, changes from IgG4), and IgG4 ProAlaAla (S228P/L234A/L235A) in its capacity to bind FcγRs and activate various immune-stimulatory responses. In contrast to the previously characterized silenced Fc variants, which retain selective FcγR binding and effector functions, IgG2σ shows no detectable binding to the Fcγ receptors in affinity and avidity measurements, nor any detectable antibody-dependent cytotoxicity, phagocytosis, complement activity, or Fc-mediated cytokine release. Moreover, IgG2σ shows minimal immunogenic potential by T-cell epitope analysis. The circulating half-life of IgG2σ in monkeys is extended relative to IgG1 and IgG2, in spite of similar in vitro binding to recombinant FcRn. The three-dimensional structure of the Fc, needed for assessing the basis for the absence of effector function, was compared with that of IgG2 revealing a number of conformational differences near the hinge region of the CH2 domain that result from the amino acid substitutions. Modeling reveals that at least one of the key interactions with FcγRs is disrupted by a conformational change that reorients P329 to a position that prevents it from interacting with conserved W90 and W113 residues of the FcγRs. Inspection of the structure also indicated significant changes to the conformation of D270 and P329 in the CH2 domain that could negatively impact C1q binding. Thus, structural perturbations of the Fc provide a rationale for the loss of function. In toto, these properties of IgG2σ suggest it is a superior alternative to previously described IgG variants of minimal effector function, for future therapeutic applications of non-immunostimulatory mAb and Fc-fusion platforms.
    Methods 07/2013; · 3.64 Impact Factor
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    ABSTRACT: Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.
    The Journal of Immunology 10/2012; · 5.52 Impact Factor
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    ABSTRACT: INTRODUCTION: Recent studies reported that human immunoglobulin G (IgG) antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2 overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody dependent cellular cytotoxicity (ADCC). The objectives of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both breast cancer cell culture method and in vivo mouse xenograft tumor model. METHODS: Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases (MMPs) and proteolytic cleavage in the lower hinge region was detected using both Western blotting and mass spectrometry. The single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate antibody dependent cellular cytotoxicity (ADCC) and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry (IHC) method. RESULTS: The single hinge cleaved trastuzumab (scIgG-T) retains HER2 antigen binding activity, inhibits HER2 mediated downstream signaling, and cell proliferation in vitro when compared with the intact trastuzumab. However, the scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry (IHC) showed reduced immune cell infiltration in tumor tissues treated with the scIgG-T when compared with these treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro. CONCLUSION: Trastuzumab can be cleaved by matrix metalloproteinases (MMP) within the lower hinge. The single hinge cleaved trastuzumab (scIgG-T) exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where MMPs often have high levels of expression and the single hinge cleaved trastuzumab might compromise its anti-tumor efficacy in clinic. However, further studies are needed to validate these hypotheses in the clinical setting.
    Breast cancer research: BCR 08/2012; 14(4):R116. · 5.87 Impact Factor
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    ABSTRACT: Immunoglobulin G (IgG) antibodies are an integral part of the adaptive immune response that provide a direct link between humoral and cellular components of the immune system. Insights into relationships between the structure and function of human IgGs have prompted molecular engineering efforts to enhance or eliminate specific properties, such as Fc-mediated immune effector functions. Human IgGs have an N-glycosylation site at Asn297, located in the second heavy chain constant region (CH2). The composition of the Fc glycan can have substantial impacts on Fc gamma receptor(FcγR) binding. The removal of the glycan through enzymatic deglycosylation or mutagenesis of the N-linked glycosylation site has been reported to "silence" FcγR-binding and effector functions, particularly with assays that measure monomeric binding. However, interactions between IgGs and FcγRs are not limited to monomeric interactions but can be influenced by avidity, which takes into account the sum of multimeric interactions between antigen-engaged IgGs and FcγRs. We show here that under in vitro conditions, which allowed avidity binding, aglycosylated IgGs can bind to one of the FcγRs, FcγRI, and mediate effector functions. These studies highlight how the valency of a molecular interaction (monomeric binding versus avidity binding) can influence antibody/FcγR interactions such that avidity effects can translate very low intrinsic affinities into significant functional outcomes.
    Journal of Molecular Recognition 03/2012; 25(3):147-54. · 3.01 Impact Factor
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    ABSTRACT: The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs, and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.
    mAbs 11/2011; 3(6):558-67. · 5.28 Impact Factor
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    Randall J Brezski, David M Knight, Robert E Jordan
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    ABSTRACT: Human anti-IgG hinge (HAH) autoantibodies constitute a class of immunoglobulins that recognize cryptic epitopes in the hinge region of antibodies exposed after proteolytic cleavage, but do not bind to the intact IgG counterpart. Detailed molecular characterizations of HAH autoantibodies suggest that they are, in some cases, distinct from natural autoantibodies that arise independent of antigenic challenge. Multiple studies have attempted to define the specificity of HAH autoantibodies, which were originally detected as binding to fragments possessing C-terminal amino acid residues exposed in either the upper or lower hinge regions of IgGs. Numerous investigators have provided information on the isotype profiles of the HAH autoantibodies, as well as correlations among protease cleavage patterns and HAH autoantibody reactivity. Several biological functions have been attributed to HAH autoantibodies, ranging from house-cleaning functions to an immunosuppressive role to restoring function to cleaved IgGs. In this review, we discuss both the historic and current literature regarding HAH autoantibodies in terms of their origins, specificity, and proposed biological relevance.
    The Scientific World Journal 01/2011; 11:1153-67. · 1.73 Impact Factor
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    Randall J Brezski, Robert E Jordan
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    ABSTRACT: The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the "nick" of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts. This suggested a pathway by which tumors might avoid host immune surveillance under a cloak of proteolytically-generated, dysfunctional antibodies that block competent IgG binding. The host immune system cannot be blind to this pathway since there exists a widespread, low-titer incidence of anti-hinge (cleavage-site) antibodies in the healthy population. The prevalence of anti-hinge reactivity may reflect an ongoing immune recognition of normal IgG catabolism. Tumor growth and bacterial infections potentially generate hostile proteolytic environments that may pose harsh challenges to host immunity. Recent findings involving physiologically-relevant proteases suggest that the potential loss of key effector functions of host IgGs may result from subtle and limited proteolytic cleavage of IgGs and that such events may facilitate the incursion of invasive cells in local proteolytic settings.
    mAbs 01/2010; 2(3):212-20. · 5.28 Impact Factor
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    ABSTRACT: The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fc gamma receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the "nick" of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fc gamma family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.
    Proceedings of the National Academy of Sciences 10/2009; 106(42):17864-9. · 9.81 Impact Factor
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    ABSTRACT: The survival of transitional and mature B cells requires both the B cell antigen receptor (BCR) and BLyS receptor 3 (BR3), which suggests that these receptors send signals that are nonredundant or that engage in crosstalk with each other. Here we show that BCR signaling induced production of the nonclassical transcription factor NF-kappaB pathway substrate p100, which is required for transmission of BR3 signals and thus B cell survival. The capacity for sustained p100 production emerged during transitional B cell differentiation, the stage at which BCR signals begin to mediate survival rather than negative selection. Our findings identify a molecular mechanism for the reliance of primary B cells on continuous BR3 and BCR signaling, as well as for the gradual resistance to negative selection that is acquired during B cell maturation.
    Nature Immunology 01/2009; 9(12):1379-87. · 26.20 Impact Factor
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    ABSTRACT: A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.
    The Journal of Immunology 10/2008; 181(5):3183-92. · 5.52 Impact Factor
  • Randall J Brezski, John G Monroe
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    ABSTRACT: The subunit structure of the B-cell antigen receptor (BCR) and its associated compartmentalization of function confer enormous flexibility for generating signals and directing these toward specific and divergent cell fate decisions. Like all the multichain immune recognition receptors discussed in this volume, assembly of these multi-unit complexes sets these receptors apart from almost all other cell surface signal transduction proteins and affords them the ability to participate in almost all of the diverse aspects of, in this case, B-cell biology. We discuss here the structural aspects of the BCR and its associated coreceptors and relate these mechanistically to how BCR signaling can be directed towards specific fate decisions. By doing so, the BCR plays a pivotal role in ensuring the effective and appropriate B-cell response to antigen.
    Advances in experimental medicine and biology 02/2008; 640:12-21. · 1.83 Impact Factor
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    Randall J Brezski, John G Monroe
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    ABSTRACT: The BCR-triggered responses of mature and transitional immature B cells differ at both the biochemical and functional level. In this study, we show that in mature B cells, BCR signaling triggers Vav phosphorylation and Rac1 activation. Furthermore, we demonstrate that although downstream actin-dependent BCR capping is independent of Rac1 activation, actin-dependent membrane ruffling and cell spreading are Rac1-dependent processes. In contrast, BCR-induced Vav phosphorylation and Rac1 activation is impaired in transitional immature B cells, resulting in defects in actin polymerization-dependent spreading and membrane ruffling while Rac1-independent BCR capping remains intact. Because transitional immature murine B cells maintain lower steady-state levels of plasma membrane cholesterol, we augmented their levels to that of mature B cells and found that BCR-induced Rac1 activation and Rac1-dependent membrane ruffling and cell spreading were restored. These studies provide a direct link between B cell cholesterol levels and downstream cellular signaling processes.
    The Journal of Immunology 11/2007; 179(7):4464-72. · 5.52 Impact Factor
  • Randall J Brezski, John G Monroe
    Advances in experimental medicine and biology 02/2007; 596:47-55. · 1.83 Impact Factor
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    ABSTRACT: Recent studies argue for an important role for cholesterol in maintaining plasma membrane heterogeneity and influencing a variety of cellular processes, including signaling, adhesion, and permeability. Here, we document that tolerance-sensitive transitional immature B cells maintain significantly lower membrane unesterified cholesterol levels than mature-stage splenic B cells. In addition, the relatively low level of cholesterol in transitional immature B cells impairs compartmentalization of their B cell receptor (BCR) into cholesterol-enriched domains following BCR aggregation and reduces their ability to sustain certain aspects of BCR signaling as compared with mature B cells. These studies establish an unexpected difference in the lipid composition of peripheral transitional immature and mature B cells and point to a determining role for development-associated differences in cholesterol content for the differential responses of these B cells to BCR engagement.
    Journal of Biological Chemistry 08/2005; 280(27):25621-8. · 4.65 Impact Factor
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    Randall J Brezski
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    ABSTRACT: B cells undergo different antigen receptor induced signaling responses and cell fate decisions depending on their stage of development. Transitional immature B cells contain less cholesterol than mature B cells, and antigen receptor stimulated transitional immature B cells fail to compartmentalize their B cell receptor (BCR) with cholesterol-enriched plasma membrane. Cholesterol is a key component in the formation of the membrane microdomain termed the lipid raft, and lipid rafts have been proposed to function as platforms for antigen receptor signal transduction. Augmentation of cholesterol levels in transitional immature B cells such that the plasma membrane has equal levels of cholesterol compared to mature B cells results in antigen receptor stimulated association of the BCR with cholesterol-enriched plasma membrane. Association of the BCR with lipid rafts correlates with sustained signaling through the PLCγ2/NFκB/c-myc pathway. In addition, mature B cells undergo directed actin polymerization following BCR stimulation, while transitional immature B cells are deficient in this response. Interestingly, Vav phosphorylation in mature B cells is stronger than transitional immature B cells, and this correlates with increased GTP-loading of Rac1. Also, BCR stimulation of mature B cells leads to membrane ruffling, a Rac1-dependent process. Transitional immature B cells do not undergo membrane ruffling following BCR stimulation consistent with the defect in Rac1-GTP loading. Cholesterol addition to transitional immature B cells rescues Rac1-GTP loading and directed actin polymerization and membrane ruffling. These studies provide a link between B cell cholesterol levels and downstream cellular signaling processes.
    Dissertations available from ProQuest. 01/2005;

Publication Stats

218 Citations
98.93 Total Impact Points

Institutions

  • 2012–2014
    • Janssen Research & Development, LLC
      Raritan, New Jersey, United States
  • 2008–2012
    • Johnson & Johnson
      New Brunswick, New Jersey, United States
    • Hospital of the University of Pennsylvania
      • Department of Pathology and Laboratory Medicine
      Philadelphia, PA, United States
  • 2005–2008
    • University of Pennsylvania
      • Department of Pathology and Laboratory Medicine
      Philadelphia, PA, United States